Anti-Histone H4 antibody [EPR21213] - ChIP Grade

• ICC

Lab

Immunocytochemistry - Anti-Histone H4 antibody [EPR21213] - ChIP Grade (AB223875)

Immunocytochemistry analysis of 100% methanol-fixed 0.1% TritonX-100 permeabilized HeLa (human cervical adenocarcinoma epithelial cell) cells staining with ab223875 at 1/100 dilution. Counterstained with ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) at 1/200 dilution. The secondary used was ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution. Confocal image showing nuclear staining in HeLa cell line (shown in green). The counterstain was observed in red. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• ICC

Lab

Immunocytochemistry - Anti-Histone H4 antibody [EPR21213] - ChIP Grade (AB223875)

Immunocytochemistry analysis of 100% methanol-fixed 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast cell) cells staining with ab223875 at 1/100 dilution. Counterstained with ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) at 1/200 dilution. The secondary used was ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution. Confocal image showing nuclear staining in NIH/3T3 cell line (shown in green). The counterstain was observed in red. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• PepArr

Lab

Peptide Array - Anti-Histone H4 antibody [EPR21213] - ChIP Grade (AB223875)

ab223875 was tested in Peptide Array against 501 different modified and unmodified histone peptides; each peptide is printed on the array at six concentrations (each in triplicate). The concentration of ab223875 was 1/500 dilution, the secondary used was Goat Anti-Rabbit IgG, (H+L), Fluo 647nm conjugated at 1/50000 dilution. Circle area represents affinity between the antibody and a peptide : all antigen-containing peptides are displayed as red circles, all other peptides as blue circles. The affinity is calculated as the area under the curve when antibody binding values are plotted against the corresponding peptide concentration. Each circle area is normalized to the peptide with the strongest affinity. The complete dataset, including a full list of all peptides and information on the position of each peptide in the diagram, can be downloaded from the Support and downloads tab.

• ChIP

Lab

ChIP - Anti-Histone H4 antibody [EPR21213] - ChIP Grade (AB223875)

Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. ChIP was performed with 25 microgram of chromatin, 2 microgram of ab223875 (red), and 20 microliter of Protein A/G sepharose beads. No antibody was added to the beads control (grey). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach). Primers and probes are located in the first kb of the transcribed region

• WB

Lab

Western blot - Anti-Histone H4 antibody [EPR21213] - ChIP Grade (AB223875)

This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% milk before being incubated with ab223875 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

All lanes:

Western blot - Anti-Histone H4 antibody [EPR21213] - ChIP Grade (ab223875) at 1 µg/mL

Lane 1:

CTH (calf Thymus Histone) at 0.5 µg

Lane 2:

HeLa Whole Cell Lysate at 20 µg

Lane 3:

HeLa Nuclear Cell Lysate at 20 µg

Lane 4:

NIH 3T3 Whole Cell Lysate at 20 µg

Lane 5:

NIH 3T3 Nuclear Lysate at 20 µg

Lane 6:

Histone H2A Recombinant Protein at 0.1 µg

Lane 7:

Histone H4 Recombinant Protein at 0.1 µg

Secondary

All lanes:

HRP conjugated Goat Anti-Rabbit IgG (H+L) at 1/50000 dilution

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Exposure time: 90s